The present invention relates to a probe for detecting polymorphism in the CYP3A gene, a method of use (e.g. a method of detecting polymorphism, a method of evaluating a drug efficacy), and a reagent kit for detecting polymorphism.
CYP (Cytochrome P450) is an oxidase involved in metabolism of a biological material such as steroid hormone and an oxidative detoxification reaction of a drug, an environmental pollutant, or the like. CYP forms a superfamily including a multitude of molecular species. Among them, CYP3A4 and CYP3A5 are known for containing polymorphism affecting metabolism of a medicine, for example (Human Mutation, 2004, Vol. 23, Issue 1, pp. 100 to 108).
The CYP3A5 gene encoding CYP3A5 is located in human chromosome 7. For example, with respect to the 401st base (r) in a partial sequence (SEQ ID NO: 1) of the CYP3A5 gene, a mutation (CYP3A5*3) from adenine (a) to guanine (g) is known (Clin. Pharmacol. Ther., 2006, Vol. 80, pp. 179 to 191, etc.). This base mutation causes an abnormal splicing, and the expression of CYP3A5 decreases greatly.
Further, the CYP3A4 gene encoding CYP3A4 is located in human chromosome 7. For example, with respect to the 201st base(s) in a partial sequence (SEQ ID NO: 2) of the CYP3A4 gene, a mutation (CYP3A4*16) from cytosine (c) to guanine (g) is known (Clin. Pharmacol. Ther., 2006, Vol. 80, pp. 179 to 191, etc.). By this base mutation, the 185th threonine (T) of CYP3A4 is mutated to serine (S).
It has been reported that these mutations have an association with, for example, metabolism of a drug such as the immunosuppressant, tacrolimus (Clin. Pharmacol. Ther., 2006, Vol. 80, pp. 179 to 191). Therefore, it is considered that there is a possibility of enabling the prediction of a tolerance to a drug with higher sensitivity by detecting these polymorphisms in the CYP3A gene.
Currently, for example, a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method is known as a method of detection polymorphism in a gene (Genet. Mol. Res., 2010, Vol. 9, Issue 1, pp. 34 to 40). In this method, PCR is performed using a primer that is designed so as to amplify a section containing a base to be detected, an amplification product is cleaved with a restriction enzyme that cleaves or not cleaves depending on the presence or absence of the mutation in the specific bases, and the determination of whether or not the section has been cleaved is performed by electrophoresis.
Further, the following method is also known. That is, in the method, a region containing mutation is amplified by a PCR method, then a melting curve analysis is performed using a nucleic acid probe labeled with a fluorescent dye, and mutation in a base sequence is analyzed based on the result of the melting curve analysis as described in JP 2002-119291 A or the like.